Alexa fluor 594 conjugated igg

Histological sections, 5 μm thick, were cut from the paraffin-embedded posterior and anterior eye samples. The sections were deparaffinized and boiled for 10 min in sodium citrate buffer (10 mM, pH 6.0; Bio-Optica, Milan, Italy) for antigen retrieval. After a pre-incubation in 1.5% bovine serum albumin (BSA) in PBS, pH 7.4 for 20 min at RT to minimize the non-specific binding, the sections were incubated overnight at 4 °C with primary antibodies directed against H1-4, diluted 1:100 in 5% BSA PBS-T (Santa Cruz Biotechnology, Dallas, TX, USA), followed by secondary Alexa Fluor 594-conjugated IgG (1:500; Jackson ImmunoResearch Europe Ltd., Cambridge House, UK).
The sections were mounted in an aqueous medium (Fluoremount, Sigma, Milan, Italy) with 4′,6-diamidino-2-phenylindole (DAPI). Representative images were acquired by an Olympus BX63 microscope coupled to CellSens Dimension Imaging Software version 1.6 (Olympus, Milan, Italy).

Immunofluorescence analysis of murine tumor mass was performed as follows. Samples were rapidly excised, fixed in buffered 4% formaldehyde for 24 h and paraffin-embedded. Histological sections, 5 μm thick, were cut from samples, were deparaffinized and boiled for 10 min in sodium citrate buffer (10 mM, pH 6.0; Bio-Optica) for antigen retrieval and immunostained over night at 4 °C with primary antibodies (anti-PD-L1, ab238697, Abcam; anti-β3-AR, ab94506, Abcam; anti-CD4, 14-9766-82, eBioscience; anti-CD8, 14-0808-82, eBioscience; DBH, ab209487, Abcam). Immune reaction was revealed incubating sections with secondary antibodies Alexa Fluor 488-conjugated IgG or Alexa Fluor 594-conjugated IgG (1:350; Jackson Laboratory). After counterstaining with 4,6-diamidino-2-phenylindole (DAPI), representative images were acquired by an Olympus BX63 microscope coupled to CellSens Dimension Imaging Software version 1.6 (Olympus). The immunofluorescence staining was evaluated through the ImageJ software (NIH, USA) as the fluorescence intensity of protein of interest was normalized to DAPI values.